Chemical display of thymine residues flipped out by DNA methyltransferases.

The DNA cytosine-C5 methyltransferase M. Hha I flips its target base out of the DNA helix during interaction with the substrate sequence GCGC. Binary and ternary complexes between M. Hha I and hemimethylated DNA duplexes were used to examine the suitability of four chemical methods to detect flipped-out bases in protein-DNA complexes. These methods probe the structural peculiarities of pyrimidine bases in DNA. We find that in cases when the target cytosine is replaced with thymine (GTGC), KMnO4proved an efficient probe for positive display of flipped-out thymines. The generality of this procedure was further verified by examining a DNA adenine-N6 methyltransferase, M. Taq I, in which case an enhanced reactivity of thymine replacing the target adenine (TCGT) in the recognition sequence TCGA was also observed. Our results support the proposed base-flipping mechanism for adenine methyltransferases, and offer a convenient laboratory tool for detection of flipped-out thymines in protein-DNA complexes.     

Dynamic modes of the flipped-out cytosine during HhaI methyltransferase-DNA interactions in solution.

Flipping of a nucleotide out of a B-DNA helix into the active site of an enzyme has been observed for the HhaI and HaeIII cytosine-5 methyltransferases (M.HhaI and M.HaeIII) and for numerous DNA repair enzymes. Here we studied the base flipping motions in the binary M. HhaI-DNA and the ternary M.HhaI-DNA-cofactor systems in solution. Two 5-fluorocytosines were introduced into the DNA in the places of the target cytosine and, as an internal control, a cytosine positioned two nucleotides upstream of the recognition sequence 5'-GCGC-3'. The 19F NMR spectra combined with gel mobility data show that interaction with the enzyme induces partition of the target base among three states, i.e. stacked in the B-DNA, an ensemble of flipped-out forms and the flipped-out form locked in the enzyme active site. Addition of the cofactor analogue S-adenosyl-L-homocysteine greatly enhances the trapping of the target cytosine in the catalytic site. Distinct dynamic modes of the target cytosine have thus been identified along the reaction pathway, which includes novel base-flipping intermediates that were not observed in previous X-ray structures. The new data indicate that flipping of the target base out of the DNA helix is not dependent on binding of the cytosine in the catalytic pocket of M.HhaI, and suggest an active role of the enzyme in the opening of the DNA duplex.     

DNA containing 4'-thio-2'-deoxycytidine inhibits methylation by HhaI methyltransferase.

4'-Thio-2'-deoxycytidine was synthesized as a 5'- protected phosphoramidite compatible with solid phase DNA synthesis. When incorporated as the target cytosine (C*) in the GC*GC recognition sequence for the DNA methyltransferase M. HhaI, methyl transfer was strongly inhibited. In contrast, these same oligonucleotides were normal substrates for the cognate restriction endonuclease R. HhaI and its isoschizomer R. Hin P1I. M. HhaI was able to bind both 4'-thio-modified DNA and unmodified DNA to equivalent extents under equilibrium conditions. However, the presence of 4'-thio-2'-deoxycytidine decreased the half-life of the complex by >10-fold. The crystal structure of a ternary complex of M. HhaI, AdoMet and DNA containing 4'-thio-2'-deoxycytidine was solved at 2.05 A resolution with a crystallographic R-factor of 0.186 and R-free of 0.231. The structure is not grossly different from previously solved ternary complexes containing M. HhaI, DNA and AdoHcy. The difference electron density suggests partial methylation at C5 of the flipped target 4'-thio-2'-deoxycytidine. The inhibitory effect of the 4'sulfur atom on enzymatic activity may be traced to perturbation of a step in the methylation reaction after DNA binding but prior to methyl transfer. This inhibitory effect can be partially overcome after a considerably long time in the crystal environment where the packing prevents complex dissociation and the target is accurately positioned within the active site.     

AdoMet-dependent methyl-transfer: Glu119 is essential for DNA C5-cytosine methyltransferase M.HhaI

The role of Glu119 in S-adenosyl-L-methionine-dependent DNA methyltransferase M.HhaI-catalyzed DNA methylation was studied. Glu119 belongs to the highly conserved Glu/Asn/Val motif found in all DNA C5-cytosine methyltransferases, and its importance for M.HhaI function remains untested. We show that formation of the covalent intermediate between Cys81 and the target cytosine requires Glu119, since conversion to Ala, Asp or Gln lowers the rate of methyl transfer 10(2)-10(6) fold. Further, unlike the wild-type M.HhaI, these mutants are not trapped by the substrate in which the target cytosine is replaced with the mechanism-based inhibitor 5-fluorocytosine. The DNA binding affinity for the Glu119Asp mutant is decreased 10(3)-fold. Thus, the ability of the enzyme to stabilize the extrahelical cytosine is coupled directly to tight DNA binding. The structures of the ternary protein/DNA/AdoHcy complexes for both the Glu119Ala and Glu119Gln mutants (2.70 A and 2.75 A, respectively) show that the flipped base is positioned nearly identically with that observed in the wild-type M.HhaI complex. A single water molecule in the Glu119Ala structure between Ala119 and the extrahelical cytosine N3 is lacking in the Glu119Gln and wild-type M.HhaI structures, and most likely accounts for this mutant's partial activity. Glu119 has essential roles in activating the target cytosine for nucleophilic attack and contributes to tight DNA binding.     

Homology modeling of the CG-specific DNA methyltransferase SssI and its complexes with DNA and AdoHcy

Prokaryotic DNA methyltransferase M.SssI recognizes and methylates C5 position of the cytosine residue within the CG dinucleotides in DNA. It is an excellent model for studying the mechanism of interaction between CG-specific eukaryotic methyltransferases and DNA. We have built a structural model of M.SssI in complex with the substrate DNA and its analogues as well as the cofactor analogue S-adenosyl-L-homocysteine (AdoHcy) using the previously solved structures of M.HhaI and M.HaeIII as templates. The model was constructed according to the recently developed "FRankenstein's monster" approach. Based on the model, amino acid residues taking part in cofactor binding, target recognition and catalysis were predicted. We also modeled covalent modification of the DNA substrate and studied its influence on protein-DNA interactions.     

Homology Modeling of the CG-specific DNA Methyltransferase SssI and its Complexes with DNA and AdoHcy

Abstract

Prokaryotic DNA methyltransferase M. SssI recognizes and methylates C5 position of the cytosine residue within the CG dinucleotides in DNA. It is an excellent model for studying the mechanism of interaction between CG-specific eukaryotic methyltransferases and DNA. We have built a structural model of M.SssI in complex with the substrate DNA and its analogues as well as the cofactor analogue S-adenosyl-L-homocysteine (AdoHcy) using the previously solved structures of M.HhaI and M.HaeIII as templates. The model was constructed according to the recently developed “FRankenstein's monster” approach. Based on the model, amino acid residues taking part in cofactor binding, target recognition and catalysis were predicted. We also modeled covalent modification of the DNA substrate and studied its influence on protein-DNA interactions.     

Cytosine methylation enhances Z-DNA formation in vivo.

The influence of cytosine methylation on the supercoil-stabilized B-Z equilibrium in Escherichia coli was analyzed by two independent assays. Both the M.EcoRI inhibition assay and the linking-number assay have been used previously to establish that dC-dG segments of sufficient lengths can exist as left-handed helices in vivo. A series of dC-dG plasmid inserts with Z-form potential, ranging in length from 14 to 74 base pairs, was investigated. Complete methylation of cytosine at all HhaI sites, including the inserts, was obtained by coexpression of the HhaI methyltransferase (M.HhaI) in cells also carrying a dC-dG-containing plasmid. Both assays showed that for all lengths of dC-dG inserts, the relative amounts of B and Z helices were shifted to more Z-DNA in the presence of M.HhaI than in the absence of M.HhaI. These results indicate that cytosine methylation enhances the formation of Z-DNA helices at the superhelix density present in E. coli. The B-Z equilibrium, in combination with site-specific base methylation, may constitute a concerted mechanism for the modulation of DNA topology and DNA-protein interactions.     

Inhibition of HhaI DNA (Cytosine-C5) methyltransferase by oligodeoxyribonucleotides containing 5-aza-2'-deoxycytidine: examination of the intertwined roles of co-factor,target, transition state structure and enzyme conformation

The presence of 5-azacytosine (ZCyt) residues in DNA leads to potent inhibition of DNA (cytosine-C5) methyltranferases (C5-MTases) in vivo and in vitro. Enzymatic methylation of cytosine in mammalian DNA is an epigenetic modification that can alter gene activity and chromosomal stability, influencing both differentiation and tumorigenesis. Thus, it is important to understand the critical mechanistic determinants of ZCyt's inhibitory action. Although several DNA C5-MTases have been reported to undergo essentially irreversible binding to ZCyt in DNA, there is little agreement as to the role of AdoMet and/or methyl transfer in stabilizing enzyme interactions with ZCyt. Our results demonstrate that formation of stable complexes between HhaI methyltransferase (M.HhaI) and oligodeoxyribonucleotides containing ZCyt at the target position for methylation (ZCyt-ODNs) occurs in both the absence and presence of co-factors, AdoMet and AdoHcy. Both binary and ternary complexes survive SDS-PAGE under reducing conditions and take on a compact conformation that increases their electrophoretic mobility in comparison to free M.HhaI. Since methyl transfer can occur only in the presence of AdoMet, these results suggest (1) that the inhibitory capacity of ZCyt in DNA is based on its ability to induce a stable, tightly closed conformation of M.HhaI that prevents DNA and co-factor release and (2) that methylation of ZCyt in DNA is not required for inhibition of M.HhaI.     

The role of Arg165 towards base flipping, base stabilization and catalysis in M.HhaI

Arg165 forms part of a previously identified base flipping motif in the bacterial DNA cytosine methyltransferase, M.HhaI. Replacement of Arg165 with Ala has no detectable effect on either DNA or AdoMet affinity, yet causes the base flipping and restacking transitions to be decreased approximately 16 and 190-fold respectively, thus confirming the importance of this motif. However, these kinetic changes cannot account for the mutant's observed 10(5)-fold decreased catalytic rate. The mutant enzyme/cognate DNA cocrystal structure (2.79 A resolution) shows the target cytosine to be positioned approximately 30 degrees into the major groove, which is consistent with a major groove pathway for nucleotide flipping. The pyrimidine-sugar chi angle is rotated to approximately +171 degrees, from a range of -95 degrees to -120 degrees in B DNA, and -77 degrees in the WT M.HhaI complex. Thus, Arg165 is important for maintaining the cytosine positioned for nucleophilic attack by Cys81. The cytosine sugar pucker is in the C2'-endo-C3'-exo (South conformation), in contrast to the previously reported C3'-endo (North conformation) described for the original 2.70 A resolution cocrystal structure of the WT M.HhaI/DNA complex. We determined a high resolution structure of the WT M.HhaI/DNA complex (1.96 A) to better determine the sugar pucker. This new structure is similar to the original, lower resolution WT M.HhaI complex, but shows that the sugar pucker is O4'-endo (East conformation), intermediate between the South and North conformers. In summary, Arg165 plays significant roles in base flipping, cytosine positioning, and catalysis. Furthermore, the previously proposed M.HhaI-mediated changes in sugar pucker may not be an important contributor to the base flipping mechanism. These results provide insights into the base flipping and catalytic mechanisms for bacterial and eukaryotic DNA methyltransferases.     

The mechanism of DNA cytosine-5 methylation. Kinetic and mutational dissection of Hhai methyltransferase

Kinetic and binding studies involving a model DNA cytosine-5-methyltransferase, M.HhaI, and a 37-mer DNA duplex containing a single hemimethylated target site were applied to characterize intermediates on the reaction pathway. Stopped-flow fluorescence studies reveal that cofactor S-adenosyl-l-methionine (AdoMet) and product S-adenosyl-l-homocysteine (AdoHcy) form similar rapidly reversible binary complexes with the enzyme in solution. The M.HhaI.AdoMet complex (k(off) = 22 s(-)1, K(D) = 6 microm) is partially converted into products during isotope-partitioning experiments, suggesting that it is catalytically competent. Chemical formation of the product M.HhaI.(Me)DNA.AdoHcy (k(chem) = 0.26 s(-)1) is followed by a slower decay step (k(off) = 0.045 s(-)1), which is the rate-limiting step in the catalytic cycle (k(cat) = 0.04 s(-)1). Analysis of reaction products shows that the hemimethylated substrate undergoes complete (>95%) conversion into fully methylated product during the initial burst phase, indicating that M.HhaI exerts high binding selectivity toward the target strand. The T250N, T250D, and T250H mutations, which introduce moderate perturbation in the catalytic site, lead to substantially increased K(D)(DNA(ternary)), k(off)(DNA(ternary)), K(M)(AdoMet(ternary)) values but small changes in K(D)(DNA(binary)), K(D)(AdoMet(binary)), k(chem), and k(cat). When the target cytosine is replaced with 5-fluorocytosine, the chemistry step leading to an irreversible covalent M.HhaI.DNA complex is inhibited 400-fold (k(chem)(5FC) = 0.7 x 10(-)3 s(-)1), and the Thr-250 mutations confer further dramatic decrease of the rate of the covalent methylation k(chem). We suggest that activation of the pyrimidine ring via covalent addition at C-6 is a major contributor to the rate of the chemistry step (k(chem)) in the case of cytosine but not 5-fluorocytosine. In contrast to previous reports, our results imply a random substrate binding order mechanism for M.HhaI.     

MethylViewer: computational analysis and editing for bisulfite sequencing and methyltransferase accessibility protocol for individual templates (MAPit) projects

Bisulfite sequencing is a widely-used technique for examining cytosine DNA methylation at nucleotide resolution along single DNA strands. Probing with cytosine DNA methyltransferases followed by bisulfite sequencing (MAPit) is an effective technique for mapping protein-DNA interactions. Here, MAPit methylation footprinting with M.CviPI, a GC methyltransferase we previously cloned and characterized, was used to probe hMLH1 chromatin in HCT116 and RKO colorectal cancer cells. Because M.CviPI-probed samples contain both CG and GC methylation, we developed a versatile, visually-intuitive program, called MethylViewer, for evaluating the bisulfite sequencing results. Uniquely, MethylViewer can simultaneously query cytosine methylation status in bisulfite-converted sequences at as many as four different user-defined motifs, e.g. CG, GC, etc., including motifs with degenerate bases. Data can also be exported for statistical analysis and as publication-quality images. Analysis of hMLH1 MAPit data with MethylViewer showed that endogenous CG methylation and accessible GC sites were both mapped on single molecules at high resolution. Disruption of positioned nucleosomes on single molecules of the PHO5 promoter was detected in budding yeast using M.CviPII, increasing the number of enzymes available for probing protein-DNA interactions. MethylViewer provides an integrated solution for primer design and rapid, accurate and detailed analysis of bisulfite sequencing or MAPit datasets from virtually any biological or biochemical system.     

Identification of an isoschizomer of the HhaI DNA methyltransferase in Mycoplasma arthritidis

The genome of Mycoplasma arthritidis strain 158 has modified cytosine residues at AGCT sequences that render the DNA resistant to digestion with the AluI restriction endonuclease. The DNA methyltransferase responsible for the base modification has previously been designated MarI. From the complete genome sequence of M. arthritidis , we identify Marth_orf138 as a candidate marI gene. Marth_orf138 was cloned in Escherichia coli and its TGA codons converted to TGG. DNA isolated from E. coli cells expressing the modified Marth_orf138 gene was degraded by the AluI nuclease, indicating that Marth_orf138 does not code for MarI. However, the DNA from E. coli was found to have acquired resistance to the restriction endonuclease HhaI. Genomic DNA from M. arthritidis was also found to be resistant to HhaI (recognizes GCGC). The M. arthritidis isoschizomer of the HhaI DNA methyltransferase, coded by Marth_orf138, is designated MarII. Transformation of M. arthritidis was not significantly affected by modification of plasmid at HhaI sites, indicating that the mycoplasma lacks a restriction endonuclease that recognizes GCGC sites.     

M.HhaI binds tightly to substrates containing mismatches at the target base.

The (cytosine-5) DNA methyltransferase M.HhaI causes its target cytosine base to be flipped completely out of the DNA helix upon binding. We have investigated the effects of replacing the target cytosine by other, mismatched bases, including adenine, guanine, thymine and uracil. We find that M.HhaI binds more tightly to such mismatched substrates and can even transfer a methyl group to uracil if a G:U mismatch is present. Other mismatched substrates in which the orphan guanine is changed exhibit similar behavior. Overall, the affinity of DNA binding correlates inversely with the stability of the target base pair, while the nature of the target base appears irrelevant for complex formation. The presence of a cofactor analog. S-adenosyl-L-homocysteine, greatly enhances the selectivity of the methyltransferase for cytosine at the target site. We propose that the DNA methyltransferases have evolved from mismatch binding proteins and that base flipping was, and still is, a key element in many DNA-enzyme interactions.     

Specificity in protein-DNA interactions: energetic recognition by the (cytosine-C5)-methyltransferase from HhaI

Sequence-specific interactions between proteins and DNA are essential for a variety of biological functions. The (cytosine-C5)-methyltransferase from HhaI (M.HhaI) specifically modifies the second base in GCGC sequences, employing a base flipping mechanism to access the target base being chemically modified. The mechanism of sequence-specific recognition of M.HhaI is not evident based on crystallographic structures, leading to the suggestion that recognition is linked to the flipping event itself, a process that may be referred to as energetic recognition. Using computational methods, it is shown that the free energy barriers to flipping are significantly higher in non-cognate versus the cognate sequence, supporting the energetic recognition mechanism. Energetic recognition is imparted by two protein "selectivity filters" that function via a "web" of protein-DNA interactions in short-lived, high energy states present along the base flipping pathway. Other sequence-specific DNA binding proteins whose function involves significant distortion of DNA's conformation may use a similar recognition mechanism.     

Engineered extrahelical base destabilization enhances sequence discrimination of DNA methyltransferase M.HhaI

Improved sequence specificity of the DNA cytosine methyltransferase HhaI was achieved by disrupting interactions at a hydrophobic interface between the active site of the enzyme and a highly conserved flexible loop. Transient fluorescence experiments show that mutations disrupting this interface destabilize the positioning of the extrahelical, "flipped" cytosine base within the active site. The ternary crystal structure of the F124A M.HhaI bound to cognate DNA and the cofactor analogue S-adenosyl-l-homocysteine shows an increase in cavity volume between the flexible loop and the core of the enzyme. This cavity disrupts the interface between the loop and the active site, thereby destabilizing the extrahelical target base. The favored partitioning of the base-flipped enzyme-DNA complex back to the base-stacked intermediate results in the mutant enzyme discriminating better than the wild-type enzyme against non-cognate sites. Building upon the concepts of kinetic proofreading and our understanding of M.HhaI, we describe how a 16-fold specificity enhancement achieved with a double mutation at the loop/active site interface is acquired through destabilization of intermediates prior to methyltransfer rather than disruption of direct interactions between the enzyme and the substrate for M.HhaI.     

Mode of action of the Spiroplasma CpG methylase M.SssI.

The cytosine DNA methylase from the wall-less prokaryote, Spiroplasma strain MQ1 (M.SssI) methylates completely and exclusively CpG-containing sequences, thus showing sequence specificity which is similar to that of mammalian DNA methylases. M.SssI is shown here to methylate duplex DNA processively as judged by kinetic analysis of methylated intermediates. The cytosine DNA methylases, M.HpaII and M.HhaI, from other prokaryotic organisms, appear to methylate in a non-processive manner or with a very low degree of processivity. The Spiroplasma enzyme interacts with duplex DNA irrespective to the presence of CpG sequences in the substrate DNA. The enzyme proceeds along a CpG-containing DNA substrate molecule methylating one strand of DNA at a time.     

Novel procedure for the detection of 5-methylcytosine.

Bisulfite converts non-methylated cytosine in DNA to uracil leaving 5-methylcytosine unaltered. In this communication, we present a new approach omitting the conventional PCR amplification step. Bisulfite-converted methylated DNA is directly sequenced. The effectiveness of the new protocol is demonstrated by using it for the detection of 5-methylation of cytosine residues introduced by three different DNA methyltransferases (M.HaeIII, M.HpaII and M.HhaI). A simple experimental system useful to determine the sequence specificity of DNA methyltransferases is also presented.     

Functional analysis of Gln-237 mutants of HhaI methyltransferase.

When the HhaI (cytosine-5) methyltransferase (M.HhaI) binds DNA it causes the target cytosine to be flipped 180 degrees out of the helix. The space becomes occupied by two amino acids, Ser-87 and Gln-237, which enter the helix from opposite sides and form a hydrogen bond to each other. Gln-237 may be involved in specific sequence recognition since it forms three hydrogen bonds to the orphan guanosine, which is the partner of the target cytosine. We have prepared all 19 mutants of Gln-237 and tested their biochemical properties. We find that mutations of this residue greatly affect the stability of the M.HhaI-DNA complex without affecting the enzyme's specificity for the target sequence. Surprisingly, all mutants retain detectable levels of enzymatic activity.     

Water-assisted dual mode cofactor recognition by HhaI DNA methyltransferase.

Energetically competent binary recognition of the cofactor S-adenosyl-L-methionine (AdoMet) and the product S-adenosyl-L-homocysteine (AdoHcy) by the DNA (cytosine C-5) methyltransferase (M.HhaI) is demonstrated herein. Titration calorimetry reveals a dual mode, involving a primary dominant exothermic reaction followed by a weaker endothermic one, for the recognition of AdoMet and AdoHcy by M.HhaI. Conservation of the bimodal recognition in W41I and W41Y mutants of M.HhaI excludes the cation-pi interaction between the methylsulfonium group of AdoMet and the pi face of the Trp(41) indole ring from a role in its origin. Small magnitude of temperature-independent heat capacity changes upon AdoMet or AdoHcy binding by M.HhaI preclude appreciable conformational alterations in the reacting species. Coupled osmotic-calorimetric analyses of AdoMet and AdoHcy binding by M.HhaI indicate that a net uptake of nearly eight and 10 water molecules, respectively, assists their primary recognition. A change in water activity at constant temperature and pH is sufficient to engender and conserve enthalpy-entropy compensation, consistent with a true osmotic effect. The results implicate solvent reorganization in providing the major contribution to the origin of this isoequilibrium phenomenon in AdoMet and AdoHcy recognition by M.HhaI. The observations provide unequivocal evidence for the binding of AdoMet as well as AdoHcy to M.HhaI in solution state. Isotope partitioning analysis and preincubation studies favor a random mechanism for M.HhaI-catalyzed reaction. Taken together, the results clearly resolve the issue of cofactor recognition by free M.HhaI, specifically in the absence of DNA, leading to the formation of an energetically and catalytically competent binary complex.     

Fine cleavage map of a small colicin E1 plasmid carrying genes responsible for replication and colicin E1 immunity.

A small plasmid (pAO2, 1 megadalton) carrying genes responsible for replication and colicin E1 immunity has been constructed from colicin E1 plasmid (A. Oka, K. Sugimoto, and M. Takanami, Proc. Mol. Biol. Jpn., p. 113-115, 1976). pAO2 DNA was cleaved into unique fragments with seven restriction endonucleases (R.HaeII,R.HaeIII,R.HapII,R.HhaI,R.AluI,R.HgaI, and R.HinfI). R.HaeII cleaved pAO2 DNA at two sites, R.HaeIII at four sites, R.HapII at nine sites, R.HhaI at eight sites, R-AluI at nine sites, R.HgaI at two sites, and R.HinfI at four sites, respectively. The order of HaeIII fragments of pAO2 was deduced from the physical map of colicin E1 plasmid previously reported (A. Oka and M. Takanami, Nature (London) 264:193-196, 1976). HapII, HhaI, and AluI fragments of pAO2 were assigned by analyzing overlapping sets of fragments arising upon digestion of individual HaeIII fragments with one of R.HapII, R.HhaI, or R.AluI, and upon their reciprocal digestion. The cleavage sites for R.HaeII, R.HgaI, and R.HinfI were localized on HapII, HhaI, and AluI fragments by combined digestion. On the basis of these data and estimates of the size of each fragment, a fine cleavage map of pAO2 was constructed.